PROJECT SUMMARY Bacterial pneumonia is a leading cause of morbidity and mortality worldwide. Increasing antimicrobial resistance among common agents of bacterial pneumonia necessitates the development of new therapeutic strategies. In this project, we focus on two resistant pathogens that are public health threats. Burkholderia pseudomallei (BP) is a common etiology of pneumonia (pneumonic melioidosis) in Southeast Asia and northern Australia. Pneumonic melioidosis is lethal in 22-50% of cases despite treatment. BP is a facultative intracellular pathogen that is inherently resistant to many antibiotics and requires prolonged courses of therapy. Klebsiella pneumoniae (KP) is an extracellular pathogen that is a well known cause of community- and healthcare-associated pneumonia. KP has become increasingly resistant to carbapenems and third generation cephalosporins. Infections caused by resistant strains of KP are difficult to treat, prolong hospital stays, and are associated with high mortality. BP and KP are representative of the urgent need to develop new therapies to treat resistant lung infections. This project brings together three investigators from distinct disciplines to tackle this challenge. Drs. West and Skerrett are established researchers in pulmonary host defense against bacterial lung infections. They have created murine models of bacterial respiratory infection including BP (and surrogate organism, B. thailandensis) infection and KP. These bacterial respiratory infection models have been used to investigate host and bacterial factors and to evaluate therapeutics. Dr. Crane, a cancer immunotherapy researcher, has developed a novel and flexible system to create genetically engineered macrophages (GEMs) to produce a range of secreted proteins over a month in vitro or in vivo. Administered intravenously to mice, GEMs accumulate at high levels in the lungs for at least 4 days. Others have reported that airway delivery of macrophages results in durable localization of these cells within the lungs for months. Thus, intravenous or pulmonary delivery of GEMs may be a novel, versatile therapeutic strategy against lung infections. The central hypothesis of this proposal is that GEMs that produce pro-inflammatory and/or antimicrobial peptides and home to the site of infection can augment host resistance to respiratory infections caused by pathogens such as BP and KP. This hypothesis will be tested as follows: Aim 1. Develop and test GEMs with enhanced bacterial killing functions that produce the cytokine interleukin 12 (IL-12) or antimicrobial peptide CRAMP (the mouse homolog of human cathelicidin). Aim 2. Define localization and the inflammatory responses induced by IL-12- or CRAMP-expressing GEMs adoptively transferred in vivo. Aim 3. Determine whether the adoptive transfer of IL-12- or CRAMP-expressing GEMs augments resistance to acute bacterial pneumonia caused by B. thailandensis or K. pneumoniae. This innovative project tests two novel and potentially synergistic therapies for resistant yet distinct pathogens causing pneumonia. Moreover, the highly adaptable and tunable GEM platform is potentially very relevant to a wide variety of other lung infections and lung diseases.